This invention relates, in part, to newly identified polynucleotides and polypeptides; variants and derivatives of the polynucleotides and polypeptides, processes for making the polynucleotides and the polypeptides, and their variants and derivatives, agonists and antagonists of the polypeptides; and uses of the polynucleotides, polypeptides, variants, derivatives, agonists and antagonists. In particular, in these and in other regards, the invention relates to polynucleotides and polypeptides of human Cystatin F.
This invention relates, in part, to newly identified polynucleotides and polypeptides; variants and derivatives of the polynucleotides and polypeptides; processes for making the polynucleotides and the polypeptides, and their variants and derivatives; agonists and antagonists of the polypeptides; and uses of the polynucleotides, polypeptides, variants, derivatives, agonists and antagonists. In particular, in these and in other regards, the invention relates to polynucleotides and polypeptides of human Cystatin F.
The cystatin superfamily comprises a group of cysteine proteinase inhibitors which are widely distributed in human tissues and body fluids, and which form tight and reversible complexes with cysteine proteinases such as cathepsins B, H, L, and S. The cystatins are most likely involved in the regulation of normal or pathological processes in which these proteinases participate. Thus, cystatins may influence the intra- and extracellular catabolism of proteins and peptides (Barret, A. J. and Kirchke, H., Methods Enzymol., 80:535-561 (1981)), regulate proteolytic processing of pro-hormones (Orlowski, M., Mol. Cell. Biochem., 52:49-74 (1983)) and pro-enzymes (Taugner, R., et al., Histochemistry, 83:103-108 (1985)), protect against penetration of normal tissues by malignant cells (Sloane, B. F., Semin. Cancer Biol., 1:137-152 (1990)) or microorganisms (Bjorck, L., et al., Nature, 337:385-386 (1989) and Bjorck, L., et al., J. Virol., 64:941-943 (1990)) and modulate local inflammatory processes in rheumatoid arthritis (Mort, J. S., et al., Arthritis Rheum., 27:509-515 (1984)) and purulent bronchiectasis (Buttle, D. J., et al., Scand. J. Clin. Lab. Invest., 50:509-516 (1990)).
The cystatin superfamily has been sub-divided into families I, II and III (also called the stefin, cystatin and kininogen families, respectively), each with members differing from those of the other families in structural organization and biological distribution (Barret, A. J., et al., Biochem. J., 236:312 (1986)). The family I cystatins A and B are small proteins consisting of single polypeptide chains of about 100 amino acid residues without disulfide bridges. The family II cystatins consist of polypeptide chains of approximately 120 amino acid residues with two intra-chain disulfide bonds. Finally, the family III cystatins, the kininogens, display a higher degree of structural complexity characterized by the presence of three family II cystatin-like domains, each with two disulfide bridges at positions homologous to those in family II cystatins (Muller-Esterl, W., et al., Transbiochem. Sci., 11:336-339 (1986)). Family I and II cystatins are mainly present intracellularly and in secretory fluids (Abrahamson, M., et al., J. Biol. Chem., 261:11282-11289 (1986)), whereas kininogens are highly concentrated in blood plasma (Adam, A., et al., Clin. Chem., 31:423-426 (1985)).
At least one type II cystatin, designated cystatin C, appears to be expressed in all tissues (Abrahamson, M., et al., Biochem. J., 268:287-294 (1990)). In contrast, S-type cystatins are found predominantly in saliva (Abrahamson, M., et al., J. Biol. Chem., 261:11282-11289 (1986)). Cystatins and derivative peptides possess antibacterial and antiviral activities (Bjorck, et al. (1989, 1990)), consistent with their presence in secretions bathing epithelial surfaces directly exposed to the environment. The cystatins may also modulate the immune response. This could occur directly, by inhibiting cysteine proteases releases by macrophages (Bieth, J., Cysteine Proteinases and Their Inhibitors, V. Turk, ed. (Walter De Gruyter and Company, New York) pp. 693-703 (1986)), or indirectly, by inhibiting the chemotaxic response and the phagocytosis-associated respiratory burst of the cells (Leung-Tack, et al., Biol. Chem., 371:255-258 (1990)). This data suggests that type II cystatins might perform a variety of protective functions at epithelial surfaces. The human type II cystatin gene family consists of at least seven members.
The disease hereditary cystatin C amyloid angiopathy (HCCAA) is associated with a Glu(copyright) Leu mutation in the gene encoding cystatin C. This leads to deposition of amyloid fibrils comprised of this mutant cystatin C in the cerebral arteries, which appears to cause fatal hemorrhaging (Ghiso, J., et al., PNAS, USA, 83:2974-2978 (1986)).
The effects of cystatin family protease inhibitors are varied and influence numerous functions, both normal and abnormal, in the biological processes of the mammalian system. There is a clear need, therefor, for identification and characterization of proteins that influence biological activity, both normally and in disease states. In particular, there is a need to isolate and characterize additional cystatins akin to known cystatins which may be employed, therefore, for preventing, ameliorating or correcting dysfunctions or disease or augmenting positive natural actions of such receptors.
Toward these ends, and others, it is an object of the present invention to provide polypeptides, inter alia, that have been identified as novel Cystatin F by homology between the amino acid sequence set out in FIG. 1 (SEQ ID NO:2) and known amino acid sequences of other proteins such as human cystatin C (SEQ ID NO:3).
It is a further object of the invention, moreover, to provide polynucleotides that encode Cystatin F, particularly polynucleotides that encode the polypeptide herein designated Cystatin F.
In a particularly preferred embodiment of this aspect of the invention the polynucleotide comprises the region encoding human Cystatin F in the sequence set out in FIG. 1 (SEQ ID NO:2).
In accordance with this aspect of the present invention there is provided an isolated nucleic acid molecule encoding a mature polypeptide expressed by the human cDNA contained in ATCC Deposit No. 97463.
In accordance with this aspect of the invention there are provided isolated nucleic acid molecules encoding human Cystatin F, including mRNAs, cDNAs, genomic DNAs and, in further embodiments of this aspect of the invention, biologically, diagnostically, clinically or therapeutically useful variants, analogs or derivatives thereof, or fragments thereof, including fragments of the variants, analogs and derivatives.
Among the particularly preferred embodiments of this aspect of the invention are naturally occurring allelic variants of human Cystatin F.
It also is an object of the invention to provide Cystatin F polypeptides, particularly human Cystatin F polypeptides, that may be employed to treat and/or prevent bacterial infection, viral infection, inflammation, protection of the eye and remodeling of the eye. Cystatin F may also be employed to regulate T-cell function and therefore regulate immune responses and may also be employed to treat immunological disorders.
In accordance with this aspect of the invention there are provided novel polypeptides of human origin referred to herein as Cystatin F as well as biologically, diagnostically or therapeutically useful fragments, variants and derivatives thereof, variants and derivatives of the fragments, and analogs of the foregoing.
Among the particularly preferred embodiments of this aspect of the invention are variants of human Cystatin F encoded by naturally occurring alleles of the human Cystatin F gene.
It is another object of the invention to provide a process for producing the aforementioned polypeptides, polypeptide fragments, variants and derivatives, fragments of the variants and derivatives, and analogs of the foregoing.
In a preferred embodiment of this aspect of the invention there are provided methods for producing the aforementioned Cystatin F polypeptides comprising culturing host cells having expressibly incorporated therein an exogenously-derived human Cystatin F-encoding polynucleotide under conditions for expression of human Cystatin F in the host and then recovering the expressed polypeptide.
In accordance with another object the invention there are provided products, compositions, processes and methods that utilize the aforementioned polypeptides and polynucleotides for research, biological, clinical and therapeutic purposes, inter alia.
In accordance with certain preferred embodiments of this aspect of the invention, there are provided products, compositions and methods, inter alia, for, among other things: assessing Cystatin F expression in cells by determining Cystatin F polypeptides or Cystatin F-encoding mRNA; assaying genetic variation and aberrations, such as defects, in Cystatin F genes; and administering a Cystatin F polypeptide or polynucleotide to an organism to augment Cystatin F function or remediate Cystatin F dysfunction.
In accordance with certain preferred embodiments of this and other aspects of the invention there are provided probes that hybridize to human Cystatin F sequences.
In certain additional preferred embodiments of this aspect of the invention there are provided antibodies against Cystatin F polypeptides. In certain particularly preferred embodiments in this regard, the antibodies are highly selective for human Cystatin F, which may be employed diagnostically to detect hereditary cystatin C amyloidosis angiopathy (HCCAA) and neoplasia.
In accordance with another aspect of the present invention, there are provided Cystatin F agonists. Among preferred agonists are molecules that mimic Cystatin F, that bind to Cystatin F-binding molecules or receptor molecules, and that elicit or augment Cystatin F-induced responses. Also among preferred agonists are molecules that interact with Cystatin F or Cystatin F polypeptides, or with other modulators of Cystatin F activities, and thereby potentiate or augment an effect of Cystatin F or more than one effect of Cystatin F.
In accordance with yet another aspect of the present invention, there are provided Cystatin F antagonists. Among preferred antagonists are those which mimic Cystatin F so as to bind to Cystatin F, receptor or binding molecules but not elicit a Cystatin F-induced response or more than one Cystatin F-induced response. Also among preferred antagonists are molecules that bind to or interact with Cystatin F so as to inhibit an effect of Cystatin F or more than one effect of Cystatin F or which prevent expression of Cystatin F. The antagonists may be used to inhibit the action of Cystatin F polypeptides, for instance, in the treatment and/or prevention of cerebral hemorrhages and encephalopathy and to inhibit HIV infection.
In a further aspect of the invention there are provided compositions comprising a Cystatin F polynucleotide or a Cystatin F polypeptide for administration to cells in vitro, to cells ex vivo and to cells in vivo, or to a multicellular organism. In certain particularly preferred embodiments of this aspect of the invention, the compositions comprise a Cystatin F polynucleotide for expression of a Cystatin F polypeptide in a host organism for treatment of disease. Particularly preferred in this regard is expression in a human patient for treatment of a dysfunction associated with aberrant endogenous activity of Cystatin F.
Other objects, features, advantages and aspects of the present invention will become apparent to those of skill from the following description. It should be understood, however, that the following description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only. Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following description and from reading the other parts of the present disclosure.